Many biological signals act via cell surface receptors and GTP-binding G proteins. Recent findings have revealed new and unexpected conformational changes in the G protein ? subunit. Our overall hypothesis is that G proteins exist in a larger ensemble of functionally-distinct structural conformations, some of which may be targeted therapeutically. We propose to investigate these changes and their consequences for signaling. The project is a collaboration between two laboratories with distinct and complementary expertise, and will combine molecular genetics (Dohlman), structural biophysics (Campbell), biochemistry and pharmacology. The proposed work is innovative because it employs, for the first time, high resolution NMR to investigate G? protein conformations and inhibitor binding interactions. The proposed work is significant because perturbations in G protein function underlie human pathologies including cardiovascular damage, infectious disease, and cancer. Aim 1. Functional analysis of G? proteins in human diseases (Dohlman). Several human cancers arise from mutations in G?. Cholera is an infectious and deadly form of diarrhea, caused by a bacterial toxin that covalently modifies G?s. In these instances the G protein is no longer able to hydrolyze GTP. We have identified a panel of second-site suppressor mutations that appear to stabilize the inactive (GDP-like) conformation, despite an inability to hydrolyze GTP. We will now compare the function of G proteins with GTPase deficient (oncogenic) mutations or that are ADP-ribosylated, in the absence or presence of our suppressor mutations. We will reconstitute G? with its known binding partners and determine how these suppressor mutations affect interactions with guanine nucleotides, G protein ?? subunits, the GTPase- activating protein, as well as recently identified protein kinases and phosphatases. Aim 2. Structural analysis of G? proteins in human diseases (Campbell). G? proteins undergo dramatic conformational changes during activation and inactivation. These changes have so far been documented by low resolution or static techniques. Our new preliminary data show the feasibility of conducting high resolution NMR on G? proteins. NMR is ideal for detecting structural and dynamic changes in proteins, including those that are transient and dynamic. We will now conduct a detailed analysis of the conformational changes imposed by mutational activation, in the absence or presence of our suppressor mutations, and in the absence or presence of small molecules that bind to G?. In the longer term, this work will reveal the structural basis for altered protein-protein interactions as well as the potential for small molecule suppressors of disease- causing mutations.